APPROXIMATING THE ISOMETRIC FORCE CALCIUM RELATION OF INTACT FROG-MUSCLE USING SKINNED FIBERS

In previous papers we used estimates of the composition of frog muscle and calculations involving the likely fixed charge density in myofibr ils to propose bathing solutions for skinned fibers, which best mimic the normal intracellular milieu of intact muscle fibers. We tested pre dictions of this calculation using measurements of the potential acros s the boundary of skinned frog muscle fibers bathed in this solution, The average potential was -3.1 mV, close to that predicted from a simp le Donnan equilibrium. The contribution of ATP hydrolysis to a diffusi on potential was probably small because addition of 1 mM vanadate to t he solution decreased the fiber actomyosin ATPase rate (measured by hi gh-performance liquid chromatography) by at least 73% but had little e ffect on the measured potential. Using these solutions, we obtained fo rce-pCa curves from mechanically skinned fibers at three different tem peratures, allowing the solution pH to change with temperature in the same fashion as the intracellular pH of intact fibers varies with temp erature. The bath concentration of Ca2+ required for half-maximal acti vation of isometric force was 1.45 mu M (22 degrees C, pH 7.18), 2.58 mu M (16 degrees C, pH 7.25), and 3.36 mu M (5 degrees C, pH 7.59). Th e [Ca2+] at the threshold of activation at 16 degrees C was similar to 1 mu M, in good agreement with estimates of threshold [Ca2+] in intac t frog muscle fibers.